The present invention relates generally to new vector plasmids and more specifically to a vector plasmid and the process for producing the same by inserting a DNA fragment containing a gene expressible in a microorganism belonging to the genus Corynebacterium or Brevibacterium into a plasmid derived from a microorganism belonging to the genus Corynebacterium or Brevibacterium. The resultant plasmid facilitates the cloning of desired genes in a host microorganism of the genus Corynebacterim or Brevibacterium.
With the advent of genetic engineering technology, it has become possible to insert a DNA fragment containing a desired gene into a vector such as plasmids and phages, and introduce the thus obtained recombinant DNA into a microbial cell whereby the microorganism inherits the gene by the help of autonomous replication of the vector.
Such technology was first established using Escherichia coli as a host microorganism. Thus, application of the technology to the production of useful metabolites by the fortification of the specific metabolic system and the production of useful proteins by the introduction of a gene derived from eucaryotes has been conducted using Escherichia coli.
Although the accumulated knowlege on Escherichia coli has greatly contributed to the progress of these researches, the successful development of useful vectors cannot be neglected. The importance of vectors in genetic engineering technology is clearly recognized in "Recombinant Molecules: Impact on Science and Society", Miles International Symposium Series No. 10, edited by R. F. Beers and E. G. Basset, Raven Press, New York, 1977.
Plasmid vectors are preferred in applied research using Escherichia coli as the host. A typical plasmid vector, pBR322, illustrates the advantages of plasmid vectors. For example pBR322 DNA can be readily recovered because of its replication characteristic of having many copies in a cell and a DNA fragment can be cloned in the plasmid without preventing the replication of pBR322 because of its small molecular weight and only one cleavage site for various restriction endonucleases. Moreover, genes responsible for ampicillin resistance and tetracycline resistance (hereinafter referred to as "Am.RTM. gene" and "Tc.RTM. gene" respectively) can be used as a marker to select a microorganism containing the plasmid.
The insertional inactivation is an additional advantage of pBR322. It has only one restriction site for each of PstI, BamHI, HindIII and SalI and the PstI site resides in the Am.RTM. gene and the other three reside in the Tc.RTM. gene. The insertion of a DNA fragment into these restriction sites will result in the loss of resistance due to the cleavage of the gene (insertional inactivation). Thus, it is possible to select a strain having a recombinant DNA by first selecting strains which are resistant to one of the drugs (Am or Tc) and then selecting those which become sensitive to the other drug due to the insertional inactivation, see, e.g., Bolivar, F. et al.: Gene, 2, 95 (1977).
A number of practical plasmid vectors for Escherichia coli have also been constructed which are similar to pBR322 in characteristics. For example, as a plasmid vector capable of the insertional inactivation of genes responsible for drug-resistance with more restriction endonucleases, plasmid pGA22 has been prepared. Since pGA22 has genes responsible for the resistance to chloramphenicol and kanamycin (hereinafter referred to as "Cm.RTM. gene" and "Km.RTM. gene" respectively) in addition to Am.RTM. gene and Tc.RTM. gene of pBR322, the insertional inactivation of Cm.RTM. gene at EcoRI site and of Km.RTM. gene at HindIII or XhoI site is possible as well as the insertional inactivation of Am.RTM. gene and Tc.RTM. gene See An. G. et al.: J. Bacteriol., 140, 400 (1979).
While, for industrially useful microorganisms other than Escherichia coli, such as amylase-producing Bacillus subtilis, antibiotics-producing Actinomycetes and alcohol-producing yeasts, recombinant DNA technology has been developed and vectors in these microorganisms have been obtained, examples of practical application of the technology using these organisms as a host are few. One of the reasons of such limited use is that in these microorganisms, a plasmid vector which is as useful as those of Escherichia coli has not been found. If Escherichia coli plasmids or fragments thereof are joined with plasmids for these organisms, they will automatically acquire the usefulness of the Escherichia coli plasmids described above. However, the successful use of drug resistance genes of Escherichia coli plasmids as vector markers has not been known in these other species. In this regard, it has been thought that although Gram positive Bacillus subtilis and Actinomycetes are procaryotes, genes of Gram negative Escherichia coli could not be expressed in these microorganisms. In fact, it has been reported that when a gene responsible for drug resistance of Escherichia coli used as a selection marker of an Escherichra coli vector plasmid is introduced into Bacillus subtilis or Actinomycetes after recombination with the plasmids of these microorganisms, the recombinant plasmid can replicate but the drug resistance gene can not be experessed, Kreft, J. et al.: Molec. Gen. Genet., 162. 59 (1978), Schottel, J. L. et al.: J. Bacteriol., 146, 360 (1981). Therefore, the results obtainable in Escherichia coli were not believed to be applicable directly to the above mentioned Gram positive microorganisms.
Thus, a need existed for application of the results of gene engineering technology in Escherichia coli to Gram positive microorganisms of the genera Corynebacterium and Brevibacterium in order to improve the efficient production of useful substances by these microorganisms. To this end, and in spite of the accepted theory that genes of Gram negative microorganisms are difficult to express in cells of Gram positive microorganisms, the present inventors have found that microorganisms of the genus Corynebacterium or Brevibacterium have an ability to express foreign genes of other microorganisms such as Escherichia coli.